The present invention relates to fragments of the Dengue virus glycoproteins prM and E which induce apoptosis and can be used as a therapeutic agent against Flavivirus infection and cancer.
Dengue (DEN) is the major arbovirus transmissible to humans in most tropical and subtropical zones. At present neither treatments nor vaccines are available to counter the disease. The infectious agent is the DEN virus, a member of the Flaviviridae family, which includes viruses that are highly pathogenic for humans, such as yellow fever virus, West Nile virus, tick-borne encephalitis viruses, Japanese encephalitis virus and hepatitis C and G viruses. The DEN virus is an enveloped virus of 40 to 60 nm diameter, whose genome is a single-stranded RNA molecule of positive polarity containing about 11000 nucleotides. The viral genome is associated with the C capsid protein to form the nucleocapsid (NC). The NC is surrounded with an envelope consisting of a double lipid layer issued from membranes of the endoplasmic reticulum (ER), in which the envelope glycoprotein E and the membrane protein M are anchored. The glycoproteins prM (precursor of protein M) and E of the viral envelope are translocated in the lumen of the ER and remain anchored to the ER membranes by their transmembrane domains (TMD) (FIG. 1A). The first stage of viral morphogenesis is non-covalent association of prM and E as a heterodimeric complex within the ER. The viral particle is probably assembled by a budding process in the ER. The provirions are carried in the vesicles, which transport them toward the plasmic membrane by passing through the Golgi complex. Cleavage of prM to M by proteases of the furine type in the trans-Golgi complex permits the virions to become fully infectious.
In vivo infection of murine neurons and of human hepatocytes by the DEN virus induces cell death by apoptosis. In vitro, the induction of the apoptotic process by infection with the DEN-1 and DEN-2 viruses have been reproduced in murine neuroblastoma cells (Neuro 2a) and in human hepatoma cells (HepG2), in human Hela cells, CHO, 293T and the primate cell line VERO. We have formulated the hypothesis that accumulation of glycoproteins of the envelope of the DEN virus in the ER would lead to a stress which induces apoptosis. In the case of human hepatomas, this stress would lead to activation of the transcription factor NF-xcexaB, which would control the expression of pro-apoptotic genes.
Apoptosis, or programmed cell death (PCD) is a type of cell death that is fundamentally distinct from degenerative death or necrosis. It is an active process of gene-directed cellular self-destruction which in some instances, serves a biologically meaningful homeostatic function. This can be contrasted to necrosis which is cell death occurring as the result of severe injurious changes in the environment of infected cells. For a general review of apoptosis, see Tomei, L. D. and Cope, F. O. Apoptosis: The Molecular Basis of Cell Death (1991) Cold Spring Harbor Press, N.Y.; Tomei, L. D. and Cope, F. O. Apoptosis II: The Molecular Basis of Apoptosis in Disease (1994) Cold Spring Harbor Press, N.Y.; and Duvall and Wyllie (1986) Immun. Today 7(4):115-119.
Morphologically, apoptosis is characterized by the rapid condensation of the cell with preservation of membranes. Synchronistically with the compaction of chromatin, several biochemical changes occur in the cell. Nuclear DNA is cleaved at the linker regions between nucleosomes to produce fragments which are easily demonstrated by agarose gel electrophoresis wherein a characteristic ladder develops.
Apoptosis has been linked to many biological processes, including embryogenesis, development of the immune system, elimination of virus-infected cells, and the maintenance of tissue homeostasis. Apoptosis also occurs as a result of human immunodeficiency virus (HIV) infection of CD4.sup.+ T lymphocytes (T cells). Indeed, one of the major characteristics of AIDS is the gradual depletion of CD4.sup.+ T lymphocytes during the development of the disease. Several mechanisms, including apoptosis, have been suggested to be responsible for the CD4 depletion. It is speculated that apoptotic mechanisms might be mediated either directly or by the virus replication as a consequence of the HIV envelope gene expression, or indirectly by priming uninfected cells to apoptosis when triggered by different agents.
Reference is made to standard textbooks of molecular biology that contain definitions and methods and means for carrying out basic techniques, encompassed by the present invention. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989) and the various references cited therein.
We have studied what viral morphogenesis in the cytotoxity of the DEN virus might mean for the murine neuronal cell. The first stages of assembly of the viral particle, in other words the heterodimeric association of the envelope glycoproteins prM and E in the lumen of the ER, were characterized in Neuro 2a cells infected by the FGA/89 strain of the DEN-1 virus (the viral sequence numbering begins at Met1 of the DEN polyprotein, FIG. 1B), or by starting from the established line N2aprM+E (a stable clone of the Neuro 2a cells), which contains cDNA coding for the two viral glycoproteins under the control of an inducible promoter (ecdysone expression system).
The expression of the recombinant glycoproteins prM and E in N2aprM+E cells causes cell death by apoptosis after 35 hours of induction. We attempted to identify the proapoptotic sequences in glycoproteins prM and E. The three-dimensional structure of protein E ectodomain of flaviviruses revealed the existence of three domains. Two predicted xcex1-helices (FGA/89 polyprotein residues 680 to 692, 710 to 727) positioned between the ectodomain (390 amino acids) and the TMD (FGA/89 polyprotein residues 737 to 775) of protein E (FIG. 2A). Little information is available on the spatial structure of protein prM. Protein M (FGA/89 polyprotein residues 206 to 280) produced by posttranslational cleavage of the glycoprotein prM in the trans Golgi network, is a non-glycosylated polypeptide of 75 amino acids composed of a predicted xcex2-sheet (FGA/89 polyprotein residues 206 to 224), a predicted xcex1-helix (FGA/89 polyprotein residues 224 to 245) and two TMDs (FGA/89 polyprotein residues 246 to 280) (FIG. 2B).
An object of the present invention is to provide a polypeptides from the Dengue virus glycoproteins which induces apoptosis.
Another object of the present invention is to provide a polynucleotide which encodes the polypeptide.
Another object of the present invention is a method of inducing apoptosis in a cell comprising administering the polypeptide to a cell.
Another object of the present invention is a method of screening for polypeptides which are capable of inducing apoptosis.
Another object of the present invention is a method of screening for molecules capable inhibiting apoptosis induced by the polypeptide from the Dengue virus glycoproteins which induces apoptosis.
Another object of the present invention is pro-apoptotic sequences in glycoproteins prM (DEN polyprotein residues 115 to 280) and E (DEN polyprotein residues 281 to 775) of the FGA/89 strain of DEN-1 virus (Genbank Data Library under accession AF226687).
Another object of the present invention is pro-apoptotic sequences of the strain DEN-2 virus Jamaica.
The invention also relates to monoclonal antibodies raised against DEN-1 and DEN-2 virus M proteins and their utilization for the prevention of disease and diagnostic purposes.